We have found that primary tumor growing at distant site induced production of TARC/CCL17 (a chemokine ligand for CCR4) in lungs. Thus, this is presumably to facilitate recruitment of CCR4+ Tregs which, as we recently reported, play a major role in regulation of T cell responses. Although the existence of CCR4+ breast cancers remained unknown, we have hypothesized that breast cancer also utilizes CCR4 in the lung metastasis. Utilizing in house developed strategies, such as chemotoxin that depletes CCR4+ cells (see AG000444-02), we demonstrated that CCR4 was indeed expressed on a portion of breast cancer cells that metastasize into into inflamed lungs producing TARC and MDC. This is a first report on the use of TARC/MDC-CCR4 axis in the metastatic spread of breast cancer. On the other hand, we also confirmed our second part of the hypothesis that primary tumor-induced production of TARC and MDC was also to recruit CCR4+ Tregs. Indeed, we demonstrate that in the absence of Tregs, the inherent capability of tumors alone to migrate into inflamed lungs was not sufficient to establish lung metastasis. Lung metastasis required an active participation of CCR4+ Tregs. The biological role of Tregs was to infiltrate lungs together with tumor cells to regulate or even kill anti-tumor NK cells. This presumably explains our finding that NK cell counts were significantly reduced in peripheral blood (PB) of both tumor-bearing mice and human patients with an advanced stage IV breast cancer. Thus, strategies that abrogate any part of this process, such as targeted inactivation of CCR4+ cells or direct depletion of Tregs, would be expected to improve the outcome of the disease. This in turn would activate both antitumor innate and adaptive immune responses through activation of NK cells and cytolytic T cells. Indeed, the treatment of tumor -bearing mice with TARC-chemotoxin significantly reduced lung metastasis of 4T1 cells through depletion of Tregs and metastasizing tumors and activating NK cells. The finding has been recently published (Olkhanud et al., 2009). Despite significant efforts, practically very little is known about mechanisms of suppressive activity exerted by Tregs. Recently, we have demonstrated that Tregs in human PBL consisted of at least two functionally distinct subsets, memory-type CCR4+Tregs and nave-type CCR4- Tregs (Baatar et al., 2007). CCR4+ Tregs also regulate resting CD8+ T cell responses without killing them or use of granzymes A and B (as others reported). In contrast, we have found that Tregs regulate T cell proliferation through a cell contact-dependent process involving FasL/Fas signaling. Recently we have found that CCR4+Tregs express and utilize lectin-type proteins, such as beta-galactoside-binding prottein (bGBP). bGBP and its dimeric lectin form Galectin-1 are immunosuppressive proteins expressed by activated immune cells, such as T cells, B cells and macrophages. Interestingly, bGBP actively participated in regulation of CD8+ T cells. It was used by Tregs to regulate TCR signaling of CD8+ T cells without induction of cell death, although bGBP is known to be cytotoxic for activated T cells. The mechanism of this process was in the capacity of bGBP to induce limited (non-processive) TCR signaling in target T cells;as it only activates Zap70, but not downstream molecules, such as ERK, Ras and PI3K. Although non-processive TCR signaling can lead to anergy, bGBP does not induce anergy of CD8+ T cells. In addition, we have found that Tregs utilize bGBP to inhibit PI3K that led in the blockage of Ras/MAPK and ERK. This presumably allows Tregs to transiently prevent activation of CD8+ T cells by self-antigens, while keeping responses to xenogeneic antigens unaffected, indicating the important biological role of bGBP in the maintenance and control of peripheral tolerance (Baatar et al., 2009).